Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury - PubMed (original) (raw)

Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury

Xiaohui Wang et al. Cardiovasc Res. 2013.

Abstract

Aims: We have reported that either toll-like receptor 4 deficiency (TLR4(-/-)) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury.

Methods and results: We constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and -8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production.

Conclusions: MicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

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Figures

Figure 1

TLR4 deficiency or TLR2 modulation with Pam3CSK4 increases the levels of miR-146a in the myocardium. (A) TLR4−/− (n = 4) and WT mice (n = 6) were subjected to myocardial ischaemia (60 min) followed by reperfusion (4 h). Sham surgery served as sham control (n = 4/group). (B) Mice were treated with and without Pam3CSK4 (50 µg/25 g body weight) 1h prior to myocardial ischaemia (60 min) followed by reperfusion (4 h) (n = 6–8/group). Sham surgical-operated mice treated with and without Pam3CSK4 served as sham control (n = 4/group). Hearts were harvested for qPCR analysis of miR-146a expression. (C) H9C2 cells were treated with or without Pam3CSK4 (1 µg/mL) 30 min before the cells were subjected to hypoxia (2 h) followed by reoxygenation (12 h). The cells were harvested for qPCR analysis of miR-146a expression. There were four duplicates. *P < 0.05 compared with indicated groups.

Figure 2

Transfection of LmiR-146a attenuated hypoxia/reoxygenation-induced cell injury and apoptosis in H9C2 cardiomyoblasts. H9C2 cardiomyoblasts were transfected with LmiR-146a or LmiR-Con. Stably transfected cell lines were selected using a Blasticidin-resistant marker. (A) The levels of miR-146a were significantly increased in LmiR-146a-transfected cells. (B_–_E) H9C2 cells and stably transfected cells were subjected to hypoxia (2 h) followed by reoxygenation (24 h) (H/R). Transfection of LmiR-146a attenuated H/R-increased LDH activity (B), decreased cell viability (C), and increased caspase-3/7 activity (D). There were four to six replicates in each group. *P < 0.05 compared with indicated groups.

Figure 3

Transfection of LmiR-146a suppresses the expression of IRAK1 and TRAF6 in H9C2 cardiomyoblasts. Western blot showed that transfection of LmiR-146a suppresses IRAK1 and TRAF6 in H9C2 cells. n = 4/group. *P < 0.05 compared with indicated groups.

Figure 4

Myocardial transfection of LmiR-146a suppresses IRAK1 and TRAF6 expression. LmiR-146a or LmiR-Con was transfected into mouse hearts through the right common carotid artery or via direct injection into heart muscle. (A) The expression of GFP carried by the lentiviral vector appeared in the myocardium (top). The levels of miR-146a were increased following LmiR-146a transfection (bottom). (B) Transfection of LmiR-146a suppresses IRAK1 and TRAF6 in the myocardium. n = 3/group. *P < 0.05 compared with indicated groups. Scale bar = 100 µm.

Figure 5

Transfection of LmiR-146a protects the myocardium from I/R injury. Mouse hearts were transfected with either LmiR-146a or LmiR-Con. Seven days after transfection, the hearts were subjected to I/R. (A) Transfection of LmiR-146a reduced myocardial infarct size. The infarct area (white) and the area at risk (red + white) from each section were measured using an image analyser. Ratios of risk area to left ventricle area (RA/LV) and infarct area to risk area (IA/RA) were calculated and are presented in the graphs. Photographs of representative heart sections are shown above. (B) Transfection of LmiR-146a attenuated I/R-induced cardiac dysfunction. Cardiac function was examined by echocardiography before I/R (Baseline) and at 3 and 7 days after I/R. n = 6–9/group. *P < 0.05 compared with indicated groups. &P < 0.05 compared with untransfected I/R control. EF, ejection fraction; FS: fractional shortening.

Figure 6

Transfection of LmiR-146a attenuates I/R-induced myocardial apoptosis. Mouse hearts were transfected with either LmiR-146a or LmiR-Con. Seven days after transfection, the hearts were subjected to myocardial ischaemia (60 min) followed by reperfusion (4 h). Myocardial apoptosis was examined by the TUNEL assay in the heart sections. (A) DAPI-stained nuclei are blue and TUNEL-positive cells show green fluorescence. The bar graph shows the per cent of apoptotic cells. (B) Transfection of LmiR-146a attenuated I/R-induced caspase-3/7 and -8 activities in the myocardium. n = 5–6/group. *P < 0.05 compared with indicated groups. Scale bar = 100 µm.

Figure 7

Transfection of LmiR-146a attenuated I/R-induced NFκB activation and inflammatory cytokine production. Mouse hearts were transfected with either LmiR-146a or LmiR-Con. Seven days after transfection, the hearts were subjected to myocardial ischaemia (60 min) followed by reperfusion (4 h). Transfection of LmiR-146a attenuated I/R-induced NF-κB binding activity (A) and TNFα and IL-1β production (B), and suppressed IRAK1 and TRAF6 in the myocardium (C). n = 5–6/group. *P < 0.05 compared with indicated groups.

Figure 8

Transfection of LmiR-146a attenuated I/R-induced neutrophil infiltration into the myocardium. Mouse hearts were transfected with either LmiR-146a or LmiR-Con. Seven days after transfection, the hearts were subjected to myocardial ischaemia (60 min) followed by reperfusion (4 h). The hearts were harvested and sectioned for immunohistochemical staining of neutrophils with anti-neutrophil antibody. The bar graph shows the numbers of neutrophils in examined fields. n = 3/group. *P < 0.05 compared with indicated groups.

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Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury - PubMed (original) (raw)